A quantitative detection kit and method for insect-resistant protein Cry1Da by enzyme-linked immunoassay

Abstract

The application belongs to the technical field of bioengineering, and discloses an enzyme-linked immunoassay quantitative detection kit and method for insect-resistant protein Cry1Da. The enzyme-linked immunoassay kit comprises a capture antibody and a detection antibody. The capture antibody is obtained by secretion of a hybridoma cell strain 7B1 with a preservation number of CGMCC No. 46851, and the detection antibody is obtained by secretion of a hybridoma cell strain 7F1 with a preservation number of CGMCC No. 46852. The enzyme-linked immunoassay kit and the detection method thereof have the characteristics of simple operation, simultaneous rapid detection of a large number of samples, high sensitivity and high specificity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically, it relates to an enzyme-linked immunosorbent assay kit and its detection method for quantitative detection of the insect-resistant protein Cry1Da in transgenic maize. Background Technology

[0002] Bacillus thuringiensis Bt Cryoblastus is a widespread Gram-positive bacterium whose secreted insect-resistant crystal proteins are currently the main biological pesticides. These insect-resistant crystal proteins are divided into two categories based on amino acid sequence similarity: Cry proteins and Cyt proteins. Cry proteins are toxic to the larvae of various harmful insects (such as Lepidoptera, Diptera, and Coleoptera), nematodes, and protozoa. Cry proteins have been transferred into crops to give them insect resistance; currently, crops with Cry protein genes mainly include corn, potatoes, and cotton. Cry1Da protein is one type of Cry protein.

[0003] The development and advancement of genetically modified (GM) technology have propelled the progress of biology. While GM foods can meet people's demands for increased yield and insect resistance, they also pose potential threats to human life. For example, certain genes introduced into the host can cause food toxicity; GM foods can produce allergens, leading to antibiotic resistance; and the nutritional value of food can be altered. Simultaneously with the research, development, and commercialization of GM foods, it is crucial to conduct comprehensive safety assessments, enable consumers to quickly distinguish between GM and natural foods, and establish appropriate methods for identifying and detecting GM components in GM foods. This will promote the safe management of agricultural GM organisms, protect the safety of humans, animals, and microorganisms, protect the ecological environment, and further advance research in agricultural GM biotechnology. The development of a Cry1Da enzyme-linked immunosorbent assay (ELISA) kit is of great significance for the rapid quantitative analysis of Cry1Da protein in GM crops or their derivatives. Summary of the Invention

[0004] The purpose of this invention is to provide an enzyme-linked immunosorbent assay kit and its detection method for quantitative detection of the insect-resistant protein Cry1Da in transgenic maize, which features simple operation, rapid simultaneous detection of a large number of samples, high sensitivity, and high specificity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for the quantitative detection of the insect-resistant protein Cry1Da in transgenic maize. The ELISA kit includes a capture antibody and a detection antibody. The capture antibody is secreted by hybridoma cell line 7B1 with accession number CGMCC No. 46851, and the detection antibody is obtained from a hybridoma cell line with accession number CGMCC No.The antibody was secreted from hybridoma cell line 7F1 of 46852; wherein, the variable region heavy chain amino acid sequence of the capture antibody is as shown in SEQ ID NO:1 (EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVATITSGGSHTHYPDSVKGRFTISRDNAKNTLDLKMSSLRSEDTAMYYCVRGVRGLYAMDYWGQGTSVTVSS), and the variable region light chain amino acid sequence is as shown in SEQ ID NO:2 (DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLTVYSCQQHYSTPYTFGGGTKLEIR); the variable region heavy chain amino acid sequence of the detection antibody is as shown in SEQ ID NO:1. NO:3 (EVQLVESGGDLVKPGGSLKVSCAASGFTFSSYAMSWVRQTPEKRLEWVATITSGGTYTHYPDSVKGRFTISRDNAKDTLYLQMSSLRSEDTAMYFCVRGVRGVYAIDYWGQGTSVTVSS), the amino acid sequence of the variable region light chain is shown in SEQ ID NO:4 (DIVMTQSHKLMSTSVGDRVSITCKASKDVSNAVAWYQQKPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCQQHYNTPYTFGGGTKMEIK); the amino acid sequence of the insect-resistant protein Cry1Da is shown in SEQ ID NO:5(SQRIEFARNQAISRLEGLSNLYKVYVRAFSDWEKDPTNPALREEMRIQFNDMNSALITAIPLFRVQNYEVALLSVYVQAANLHLSILRDVSVFGERWGYDTATINNRYSDLTSLIHVYTNHCVDTYNQGLRRLEGRFLSDWIVYNRFRRQLTISVLDIVAFFPNYDIRTYPIQTATQ LTREVYLDLPFINENLSPAASYPTFSAAESAIIRSPHLVDFLNSFTIYTDSLARYAYWGGHLVNSFRTGTTNLIRSPLYGREGNTERPVTITASPSVPIFRTLSYITGLDNSNPVAGIEGVEFQNTISRSIYRKSGPIDSFSELPPQDASVSPAIGYSHRLCHATFLERISGPRIAGT). .

[0006] Furthermore, the detection antibody is a biotin-labeled detection antibody.

[0007] Furthermore, the enzyme-linked immunosorbent assay kit also includes a microplate, sample processing solution, biotin-labeled detection antibody, Cry1Da anti-insect protein standard, horseradish peroxidase-labeled avidin, biotin-labeled detection antibody working solution, horseradish peroxidase-labeled avidin working solution, washing solution, substrate solution, and stop solution.

[0008] Secondly, the present invention provides a method for quantitatively detecting the insect-resistant protein Cry1Da in transgenic maize using the above-mentioned enzyme-linked immunosorbent assay kit, comprising the following steps: 1) Detection: Set up wells for the insect-resistant protein Cry1Da standard, the test sample, and the blank control. Add capture antibody to each well and incubate. Then discard the liquid and dry the well. Add the insect-resistant protein Cry1Da standard, the test sample, or the sample extraction buffer to each well and incubate. Then discard the liquid and dry the well. Add biotin-labeled detection antibody working solution to each well and incubate. Discard the liquid in the well, wash and dry the well. Add horseradish peroxidase-labeled avidin working solution to each well and incubate. Discard the liquid in the well, wash and dry the well. Add substrate solution to each well. After developing color in the dark, add stop solution to each well and mix well. After the reaction is terminated, measure the optical density (OD) value of each well using a microplate reader. 2) Data Processing: The corrected OD values ​​for each standard well were obtained by subtracting the OD value of the blank control S0 well from the OD value of each standard well. A working curve was plotted using these corrected OD values ​​to represent the concentration of the insecticidal protein Cry1Da standard added to each standard well. The corrected OD values ​​for each test sample well were then substituted into the working curve to calculate the concentration of the insecticidal protein Cry1Da in each test sample well. Furthermore, prior to the detection in step 1), each solution in the enzyme-linked immunosorbent assay kit is placed at 18-25°C and equilibrated for at least 30 minutes.

[0009] Furthermore, the terminating solution is a 1M sulfuric acid solution.

[0010] Furthermore, the optical density (OD) value of each well was measured using an ELISA reader at a wavelength of 450 nm.

[0011] Furthermore, the temperature for each incubation in each well was 37°C.

[0012] Furthermore, the working solution of the biotin-labeled detection antibody is obtained by diluting the biotin-labeled detection antibody solution with biotin-labeled antibody diluent at a ratio of 1:100, such as adding 990 μL of biotin-labeled antibody diluent to 10 μL of biotin-labeled antibody, mixing gently, and preparing within 10 minutes before use.

[0013] Furthermore, the working solution of horseradish peroxidase-labeled avidin is obtained by diluting horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100. For example, 10 μL of horseradish peroxidase-labeled avidin is added to 990 μL of horseradish peroxidase-labeled avidin diluent, and the mixture is gently mixed and prepared within 10 minutes before use.

[0014] Further, in step 1), add 80-120 μL of standard, test sample, or sample extraction buffer to each well, gently shake to mix, cover with a plate patch, and incubate at 37°C for 1.5-2.5 hours; discard the liquid, agitate dry, and do not wash; add 80-120 μL of biotin-labeled antibody working solution to each well, cover with a new plate patch, and incubate at 37°C for 0.5-1.5 hours; discard the liquid in the wells, agitate dry, wash the plate 2-4 times, soaking for 1-3 minutes each time, 150-250 μL / well, and agitate dry; add 80-120 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate patch, and incubate at 37°C for 0.5-1.5 hours; discard the liquid in the wells, agitate dry, wash the plate 4-6 times, soaking for 1-3 minutes each time, 150-250 μL / well, and agitate dry; sequentially add 80-100 μL of substrate solution to each well, incubate at 37°C... Incubate at ℃ in the dark for 15-30 minutes to develop color; then add 50 μL of stop solution to each well sequentially to terminate the reaction.

[0015] Further, in step 1), add 100 μL of standard, test sample, or sample extraction buffer to each well, gently shake to mix, cover with a plate patch, and incubate at 37 ℃ for 2 hours; discard the liquid, spin dry, and do not wash; add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate patch, and incubate at 37 ℃ for 1 hour; discard the liquid in the well, spin dry, wash the plate 3 times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate patch, and incubate at 37 ℃ for 1 hour; discard the liquid in the well, spin dry, wash the plate 5 times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 90 μL of substrate solution to each well in sequence.

[0016] Furthermore, the detection method is the ELISA double-antibody sandwich method.

[0017] Thirdly, the present invention also provides a hybridoma cell line for quantitative detection of the insect-resistant protein Cry1Da in transgenic maize, including hybridoma cell line 7B1 with accession number CGMCC No.46851 and hybridoma cell line 7F1 with accession number CGMCC No.46852, wherein the hybridoma cell line is used to secrete a monoclonal antibody that specifically recognizes the insect-resistant protein Cry1Da in transgenic maize; The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by the hybridoma cell line 7B1 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by the hybridoma cell line 7F1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the insect-resistant protein Cry1Da is shown in SEQ ID NO:5.

[0018] The hybridoma cell line 7B1 provided by this invention was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46851.

[0019] The hybridoma cell line 7F1 provided by this invention was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46852.

[0020] Compared with the prior art, the present invention has the following advantages: The kit of the present invention is a detection kit based on the ELISA double antibody sandwich method. It is low in cost, and the main reagents are provided in the form of working solution. It is easy to operate and can detect a large number of samples at the same time and quickly. It has the characteristics of high sensitivity and high specificity. It is also the first kit in China and even internationally that can quantitatively and specifically detect the insect-resistant protein Cry1Da in transgenic maize. Attached Figure Description

[0021] Figure 1 The standard curve was established for the enzyme-linked immunosorbent assay (ELISA) method provided by this invention.

[0022] Figure 2 The monoclonal antibody produced by hybridoma cell line 7B1 provided by this invention is a capture antibody, and the monoclonal antibody produced by hybridoma cell line 7F1 is a detection antibody, which are commonly used for specific detection. BtImage of sandwich ELISA detection results for Cry insect-resistant protein. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: Quantitative detection of insect-resistant protein Cry1Da in transgenic maize using an enzyme-linked immunosorbent assay kit 1. A quantitative detection method for the insect-resistant protein Cry1Da in transgenic maize (its amino acid sequence is shown in SEQ ID NO:5, specifically: SQRIEEFARNQAISRLEGLSNLYKVYVRAFSDWEKDPTNPALREEMRIQFNDMNSALITAIPLFRVQNYEVALLSVYVQAANLHLSILRDVSVFGERWGYDTATINNRYSDLTSLIHVYTNHCVDTYNQGLRRLEGRFLSDWIVYNRFRRQLTISVLDIVAFFPNYDIRTYPIQTATQLTREVYLDLPFINENLSPAASYPTFSAAESAIIRSPHLVDFLNSFTIYTDSLARYAYWG) The enzyme-linked immunosorbent assay (ELISA) kit (GHLVNSFRTGTTTNLIRSPLYGREGNTERPVTITASPSVPIFRTLSYITGLDNSNPVAGIEGVEFQNTISRSIYRKSGPIDSFSELPPQDASVSPAIGYSHRLCHATFLERISGPRIAGT) consists of a microplate coated with capture antibody, sample processing solution, biotin-labeled detection antibody, Cry1Da anti-insect protein standard, horseradish peroxidase-labeled avidin, biotin-labeled detection antibody dilution, horseradish peroxidase-labeled avidin dilution, concentrated wash buffer, substrate solution, stop solution, and plate patches.

[0027] The formulation of the sample processing solution is shown in Table 1.

[0028] Table 1

[0029] Biotin-labeled detection antibody: 100 μg / mL biotin-labeled Cry1Da 7F1 mouse monoclonal antibody (PBS, 50 v / v glycerol) solution; Standard: 12.5 ng of His-Cry1Da recombinant protein (lyophilized powder). The His-Cry1Da recombinant protein is produced by amplifying and sequencing the Cry1Da encoding gene, identifying it correctly, ligating it to the pET28a plasmid, and then expressing it using the recombinant expression vector pET28a- Cry1Da (84-440aar) The recombinant protein Cry1Da (84-440aar) expression strain was revived and cultured after being transformed into Escherichia coli BL21(DE3) competent cells. The protein expression was induced overnight at 16°C with IPTG and then purified by recombinant protein. Horseradish peroxidase-labeled avidin: 1:40 horseradish peroxidase-labeled avidin stock solution (Kinkairui Company), working solution diluted 1:100; The formulations of biotin-labeled antibody diluent / horseradish peroxidase-labeled avidin diluent are shown in Table 2.

[0030] Table 2

[0031] Concentrated washing buffer: 10×PBS containing 1.0wt% Tween-20; Substrate solution: Mix 0.5 mL of 2 mg / mL TMB anhydrous ethanol solution, 10 mL of substrate buffer, and 32 μL of 30% H2O2. Prepare fresh before use. Termination solution: 1M H2SO4.

[0032] Characteristics of the kit: 1) Sensitivity: 0.421 ng / mL 2) Precision: Intra-batch variation CV% < 8%, Inter-batch variation CV% < 10%. 3) Specificity: This kit specifically detects Cry1Da and has no cross-reactivity with other related proteins.

[0033] The capture antibody was secreted by hybridoma cell line 7B1 with accession number CGMCC No.46851. The detection range of 7B1 as a capture antibody is 1.5625 - 12.5 ng / mL.

[0034] The detection antibody was secreted by hybridoma cell line 7F1 with accession number CGMCC No. 46852.

[0035] The hybridoma cell lines 7B1 and 7F1 were obtained by immunizing BALB / c mice with the insect-resistant protein Cry1Da from transgenic maize, then fusing the spleen cells of the immunized mice with commercially available mouse hybridoma cells SP2 / 0, and screening with HAT medium.

[0036] The specific testing method includes the following steps: 1) Place each solution in the enzyme-linked immunosorbent assay kit at 18-25 ℃ and equilibrate for at least 30 minutes; 2) Set up separate wells for the Cry1Da anti-insect protein standard, the test sample, and the blank control. Add capture antibody to each well and incubate. Then discard the liquid, and after drying, add the Cry1Da anti-insect protein standard, the test sample, or the sample extraction buffer to each well and incubate. Then discard the liquid, and after drying, add biotin-labeled detection antibody working solution to each well and incubate. Discard the liquid in the well, wash and dry. Then add horseradish peroxidase-labeled avidin working solution to each well and incubate. Discard the liquid in the well, wash and dry. Then add substrate solution to each well, and after developing color in the dark, add 1M sulfuric acid solution as a stop solution to each well. Mix well. After the reaction is terminated, measure the optical density (OD) value of each well at a wavelength of 450nm using an ELISA reader. 3) Data processing: Subtract the OD value of the blank control S0 well from the OD value of each standard well to obtain the corrected OD value of each standard well. Use the corrected OD values ​​of each standard well to plot the concentration of the insecticidal protein Cry1Da standard added to each standard well. Substitute the corrected OD values ​​of each test sample well obtained by subtracting the OD value of the blank control S0 well from the OD value of each test sample well into the working curve to calculate the concentration of the insecticidal protein Cry1Da in each test sample well.

[0037] in: The working solution of biotin-labeled detection antibody is obtained by diluting the biotin-labeled detection antibody solution with biotin-labeled antibody diluent at a ratio of 1:100. For example, add 10 μL of biotin-labeled antibody to 990 μL of biotin-labeled antibody diluent, mix gently, and prepare within 10 minutes before use.

[0038] Concentrated detergent solution: Salt precipitation may occur when the detergent solution is stored at low temperatures. It can be warmed in a water bath to aid dissolution during dilution. Working solution: Dilute the concentrated detergent solution with deionized water at a ratio of 1:25. For example, measure 240 mL of deionized water using a graduated cylinder, pour it into a beaker or other clean container, then measure 10 mL of the concentrated detergent solution, add it evenly, stir well, and prepare immediately before use.

[0039] Horseradish peroxidase-labeled avidin working solution is obtained by diluting horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100. For example, add 10 μL of horseradish peroxidase-labeled avidin to 990 μL of horseradish peroxidase-labeled avidin diluent, mix gently, and prepare within 10 minutes before use.

[0040] In step 2), add 100 μL of standard, test sample, or sample extraction buffer to each well, gently shake to mix, cover with a plate patch, and incubate at 37 °C for 2 hours; discard the liquid, spin dry, and do not wash; add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate patch, and incubate at 37 °C for 1 hour; discard the liquid in the well, spin dry, wash the plate 3 times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate patch, and incubate at 37 °C for 1 hour; discard the liquid in the well, spin dry, wash the plate 5 times, soaking for 2 minutes each time, 200 μL / well, and spin dry; add 90 μL of substrate solution to each well in sequence.

[0041] The standard was diluted from 25 ng to a series of concentrations. Following the detection method described in this invention, the standard was added to each well and incubated. The liquid was then discarded. Biotin-labeled detection antibody working solution was added and incubated. The liquid in the wells was discarded, and after drying, horseradish peroxidase-labeled avidin working solution was added to each well and incubated. The liquid in the wells was discarded, and after drying, substrate solution was added to each well. After color development in the dark, stop solution was added to each well. Within 5 minutes after the reaction was terminated, the optical density (OD) values ​​of each well were measured sequentially at 450 nm using a microplate reader to create a standard curve. See details below. Figure 1 R of the standard curve 2 With a value >0.99, the linear detection range is 1.5625 - 12.5 ng / mL, leading to the formula for calculating sample concentration: x = (y + 0.017) / 0.089.

[0042] Transgenic maize leaves were collected, ground with liquid nitrogen, and thoroughly mixed with sample extraction buffer. After standing on ice for 30 minutes, the supernatant was collected, appropriately diluted, and added to each well according to the detection method described in this invention for incubation. The liquid was then discarded, and biotin-labeled detection antibody working solution was added for incubation. The liquid in the wells was discarded, and after drying, horseradish peroxidase-labeled avidin working solution was added to each well for incubation. The liquid in the wells was discarded, and after drying, substrate solution was added to each well. After color development in the dark, stop solution was added to each well. Within 5 minutes after the reaction was terminated, the optical density (OD) value of each well was measured sequentially at a wavelength of 450 nm using an ELISA reader. The Cry1Da content in the transgenic maize leaves was calculated to be x = 878.00 ± 15.27 ng / mL.

[0043] Key points of operation: 1. To ensure the accuracy of the test results, both the standard and the sample are tested in two wells, and a standard curve must be prepared for each test.

[0044] 2. If the content of the analyte in the sample is too high, it needs to be diluted with sample diluent first to make the sample conform to the detection range of the kit, and then multiplied by the corresponding dilution factor when calculating.

[0045] 3. Sample addition: Use disposable clean pipette tips to avoid cross-contamination. Add the sample gently to avoid foaming, placing it at the bottom of the wells; do not add it along the well walls. Ideally, each sample addition should be completed within 10 minutes. If there are many samples, a multi-channel pipette can be used.

[0046] 4. Incubation: To prevent sample evaporation or contamination, the ELISA plate must be covered with a plate patch during incubation. The ELISA plate should not be allowed to dry out during the experiment. The incubator temperature should be monitored regularly during incubation to ensure it remains constant at 37°C, and adjusted as needed. The incubator should not be opened too frequently during incubation to avoid affecting temperature balance.

[0047] 5. Washing: The washing process is very important; insufficient washing can easily lead to false positives.

[0048] (1) Manual plate washing method: Aspirate (without touching the walls and bottom of the wells) or shake off the liquid in the microplate; place several layers of absorbent paper on the lab bench, and tap the microplate face down several times; inject the recommended washing buffer into the wells at 200 μL / well and soak for 2 minutes. Repeat this process several times as described in the operating procedure.

[0049] (2) Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experimental process only after you are proficient in its use.

[0050] 6. Color Development: To ensure the accuracy of experimental results, the stop solution should be added as soon as the substrate reaction time is up. The color development can be observed periodically after adding the substrate solution to control the reaction time (e.g., every 10 minutes). When a clear gradient of blue is visible in the first 3-4 wells of the standard, while the color development is less obvious in the last 3-4 wells, the stop solution can be added to terminate the reaction. At this point, the blue color will immediately turn yellow. The order of adding the stop solution should ideally be the same as the order of adding the substrate solution.

[0051] 7. The substrate solution should be light blue or colorless. If the color darkens significantly, it must be discarded. The substrate solution is easily contaminated and should be stored properly away from light.

[0052] Example 2: Validation of antibody specificity using an enzyme-linked immunosorbent assay (ELISA) kit Besides Cry1Da, other commonly used Cry insect-resistant proteins in genetically modified crops include Cry1Fa and Cry1Ah. BtProtein. To detect the specificity of the monoclonal antibody produced by the hybridoma cell line 7B1 of the present invention, a sandwich ELISA was performed using monoclonal antibody (7B1) as the capture antibody and another monoclonal antibody (7F1) as the detection antibody. Cry1Ah (HGK60) and turn Cry1Ab + Cry1Fa (BFL4-2) Gene-resistant insect-resistant maize material.

[0053] The results showed that ( Figure 2 For the Cry1Da gene-transformed material, the detection wells showed a significant positive reaction, with OD450 values ​​of 0.65645 and 0.67605, respectively. In contrast, the OD450 values ​​of the Cry1Ah gene-transformed material (HGK60) were only 0.0726 and 0.0786, and the OD450 values ​​of the Cry1Ab+Cry1Fa gene-transformed material (BFL4-2) were only 0.0336 and 0.0746, both close to 0.

[0054] The above results show that the bispecific antibody (7B1 / 7F1) prepared in this invention can specifically recognize the Cry1Da protein, while showing no obvious cross-reactivity with Cry1Ah, Cry1Ab and Cry1Fa proteins, proving that the antibody has highly specific antigen-binding activity against Cry1Da.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for the quantitative detection of the insect-resistant protein Cry1Da in transgenic maize, the ELISA kit comprising a capture antibody and a detection antibody, characterized in that, The capture antibody is secreted by hybridoma cell line 7B1 with accession number CGMCC No. 46851, and the detection antibody is secreted by hybridoma cell line 7F1 with accession number CGMCC No. 46852. The amino acid sequence of the heavy chain variable region of the capture antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2. The amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of the insect-resistant protein Cry1Da is shown in SEQ ID NO:

5.

2. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The detection antibody is a biotin-labeled detection antibody.

3. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 2, characterized in that, The enzyme-linked immunosorbent assay kit also includes a microplate, sample processing solution, biotin-labeled detection antibody, Cry1Da anti-insect protein standard, horseradish peroxidase-labeled avidin, biotin-labeled detection antibody working solution, horseradish peroxidase-labeled avidin working solution, washing solution, substrate solution, and stop solution.

4. A method for quantitatively detecting the insect-resistant protein Cry1Da in transgenic maize using the enzyme-linked immunosorbent assay kit according to claim 3, characterized in that, Includes the following steps: 1) Detection: Set up wells for the insect-resistant protein Cry1Da standard, the test sample, and the blank control. Add capture antibody to each well and incubate. Then discard the liquid and dry the well. Add the insect-resistant protein Cry1Da standard, the test sample, or the sample extraction buffer to each well and incubate. Then discard the liquid and dry the well. Add biotin-labeled detection antibody working solution to each well and incubate. Discard the liquid in the well, wash and dry the well. Add horseradish peroxidase-labeled avidin working solution to each well and incubate. Discard the liquid in the well, wash and dry the well. Add substrate solution to each well. After developing color in the dark, add stop solution to each well and mix well. After the reaction is terminated, measure the optical density (OD) value of each well using a microplate reader. 2) Data processing: Subtract the OD value of the blank control S0 well from the OD value of each standard well to obtain the corrected OD value of each standard well. Use the corrected OD value of each standard well to plot the working curve of the concentration of the insecticidal protein Cry1Da standard added to each standard well. Substitute the OD value of the blank control S0 well into the corrected OD value of each test sample well, and then substitute the corrected OD value of each test sample well into the working curve to calculate the concentration of the insect-resistant protein Cry1Da in each test sample well.

5. The method according to claim 4, characterized in that, Before the detection in step 1), each solution in the enzyme-linked immunosorbent assay kit should be placed at 18-25 °C and equilibrated for at least 30 minutes.

6. The method according to claim 4, characterized in that, The terminating solution is a 1M sulfuric acid solution.

7. The method according to claim 4, characterized in that, The optical density (OD) value of each well was measured at a wavelength of 450 nm using an ELISA reader.

8. The method according to claim 4, characterized in that, The temperature for each incubation in each well is 37°C.

9. A hybridoma cell line for quantitative detection of the insect-resistant protein Cry1Da in transgenic maize, characterized in that, This includes hybridoma cell line 7B1 with accession number CGMCC No.46851 and hybridoma cell line 7F1 with accession number CGMCC No.46852, which are used to secrete monoclonal antibodies that specifically recognize the insect-resistant protein Cry1Da in transgenic maize. The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by the hybridoma cell line 7B1 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by the hybridoma cell line 7F1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the insect-resistant protein Cry1Da is shown in SEQ ID NO:5.

Images